1. PROJECT SUMMARY Our long-term research goal is to understand the function-evolution relationship in cis-regulatory modules (CRMs) such as enhancers and promoters. The main goal of this proposal is to test the hypothesis that CRMs with pleisiomorphic/ancestral activities represent essential CRMs for gene expression. This hypothesis focuses on conservation of molecular function to identify functionally important CRMs. Our preliminary quantitative analyses have shown the prevalence of CRMs that can drive reporter expression in distantly related species. Building upon the preliminary results and harnessing the power of our high-throughput methods for CRM analysis, this project will examine the necessity of CRMs with ancestral activities for expression of genes in sea urchin embryos. Aim 1 will identify CRMs with ancestral spatial activities by testing an unbiased set of 5,000 CRMs from the purple sea urchin, Strongylocentrotus purpuratus, in two distantly related echinoderms: the pencil urchin Eucidaris tribuloides and the sea star Patiria miniata. Because there is virtually no detectable sequence homology in non-coding regions between the purple urchin and the other species, trans-species CRM activities detected in this analysis are presumably ancestral. Aim 2 will examine the necessity of CRMs for gene expression in the purple urchin embryos by two complementary approaches: deletion of a select set of 30 CRMs by CRISPR, and massive perturbation of CRMs by knock-down of key regulatory genes. We will compare the results from Aim 1 and Aim 2 to test our central hypothesis. We will also test whether the observed prevalence of trans-species CRM activities is due to evolutionary constraints on CRMs and gene regulatory circuits. This project fulfills the goals of AREA program by primarily involving undergraduate and graduate students in research, specifically in functional and evolutionary analyses of CRMs that might be important for early embryonic development and related diseases. We anticipate that findings from this project can serve as an empirical foundation for rapidly identifying functionally important CRMs and related gene regulatory networks. This research may also lead to a general approach for using functional conservation rather than sequence conservation to study function-evolution relationships over almost any timescale without being limited by an inability to detect sequence homology.